Polynucleotides and Polypeptides Encoding Receptors

ABSTRACT

Receptor polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing receptor polypeptides and polynucleotides in the design of protocols for the treatment of diseases and diagnostic assays for such conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 12/431,986,filed Apr. 29, 2009, which is a continuation of application Ser. No.11/832,019, filed Aug. 1, 2007 (now abandoned), which is a continuationof application Ser. No. 11/041,419, filed Jan. 25, 2005 (now abandoned),which is a continuation of application Ser. No. 10/156,136, filed May29, 2002 (now abandoned), which is a continuation of application Ser.No. 09/764,452, filed Jan. 19, 2001 (now abandoned), which is acontinuation of application Ser. No. 09/010,146, filed Jan. 21, 1998(now abandoned), which claims the benefit under 35 U.S.C. §119(e) ofprovisional Application No. 60/034,204, filed Jan. 21, 1997 andprovisional Application No. 60/034,205, filed Jan. 21, 1997; each ofwhich is hereby incorporated by reference in its entirety.

STATEMENT UNDER 37 C.F.R. §1.77(b)(5)

This application refers to a “Sequence Listing” listed below, which wasprovided as a text document in U.S. application Ser. No. 12/431,986,filed Apr. 29, 2009, entitled “PF354C5_SequenceList.txt”. Applicantsrequest the use of the computer readable “Sequence Listing” filed inconnection with U.S. application Ser. No. 12/431,986 on Apr. 29, 2009,as the computer readable form for the instant application. Applicantshereby state that the paper copy of the “Sequence Listing” filed in theinstant application on Nov. 23, 2010, is identical to the computerreadable copy filed on Apr. 29, 2009, in U.S. application Ser. No.12/431,986, and is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Receptor proteins are found on the membrane of the cells and aregenerally involved in signal transduction. There are many types ofreceptor proteins, and for convenience, these proteins are grouped infamilies based on similarity in structure and function.

Receptor proteins are found on the membrane of the cells and aregenerally involved in signal transduction. There are many types ofreceptor proteins, and for convenience, these proteins are grouped infamilies based on similarity in structure and function.

For example, the TM4SF superfamily of cell surface proteins, also knownas the tetraspan receptor superfamily, is comprised of at leastseventeen individual gene products (these include CD9, CD20, CD37, CD53,CD63, CD81, CD82, A15, CO-029, Sm23, RDS, Uro B, Uro A, SAS, Rom-1,PETA3, and YKK8). The TM4SF superfamily is the second largest group inthe CD antigen superfamily. Each member of the TM4SF superfamily can becharacterized by several putative physical features including fourhighly conserved transmembrane domains, two divergent extracellularloops, and two short and highly divergent cytoplasmic tails. Expressionpatterns for members of the TM4SF superfamily tend to be rather broadand can vary widely between members. The functional roles of TM4SFsuperfamily members are primarily associated with signal transductionevents and pathways, but also include cell adhesion in platelets andother lymphocytic and non-lymphocytic cell lines, as well as cellmotility, proliferation, and metastasis. In addition, recent evidencesuggests that a subset of the members of the TM4SF superfamily mayfunction as potassium channel molecules.

One member of the TM4SF family, CD20, is a four membrane spanning domaincell surface phosphoprotein expressed exclusively on B lymphocytes.Although the precise functional role of CD20 has yet to be determined,it is thought to function primarily as a receptor during B-cellactivation. Furthermore, a large number of experimental observationssuggest several additional speculative roles for the CD20 molecule. Forexample, CD20-specific immunoprecipitation of biochemically cross-linkedplasma membrane proteins suggests that CD20 assumes a multimericstructural conformation characteristic of other previously describedmembrane channel proteins. Further experimentation has revealed thatexpression of exogenous CD20 on the cell surface specifically increasesCa²⁺ conductance across the plasma membrane. Together, these resultssuggest that CD20 complexes may function as B-cell specific Ca²⁺ ionchannels. In addition, monoclonal antibodies raised against CD20 havebeen used to stimulate resting B-cells to transition out of the G0/G1segment of the cell cycle. It has also been demonstrated that CD20 isassociated with both serine and tyrosine kinases and, more specifically,that CD20 is associated, although not directly, with the Src family oftyrosine kinases including p56/53lyn, p56lck, and p59fyn.

A second example of a receptor subfamily, called sialoadhesin molecules,belongs to the Ig superfamily of receptor-like molecules. The more than100 members of the Ig superfamily are generally considered to engage inspecific cell-cell interactions through which intercellularcommunication may occur. In addition to classical protein-proteininteractions, intercellular communication may also be mediated throughprotein-carbohydrate interactions. In fact, all members of thesialoadhesin family of the Ig superfamily are capable of mediatingprotein-sialic acid binding interactions. To date, only a small numberof proteins have been assigned to the sialoadhesin family includingsialoadhesin, CD33, CD22, the myelin-associated glycoprotein (MAG), andthe Schwann cell myelin protein (SMP). Each of these proteins isexpressed in a restricted subset of cell types. For example, CD22 andCD33 are expressed exclusively by B-lymphocytes and cells of themyelomonocytic lineage, respectively.

Similarly, galectins are a family of the lectin superfamily ofcarbohydrate-binding proteins which have a high affinity forb-galactoside sugars. Although a large number of glycoproteinscontaining b-galactoside sugars are produced by the cell, only a fewwill bind to known galectins in vitro. Such apparent binding specificitysuggests a highly specific functional role for the galectins. Galectin 1(conventionally termed LGALS1 for lectin, galactoside-binding,soluble-1) is thought to specifically bind laminin, a highlypolylactosaminated cellular glycoprotein, as well as the highlypolylactosaminated lysosome-associated membrane proteins (LAMPs).Galectin 1 has also been shown to bind specifically to alactosamine-containing glycolipid found on olfactory neurons and tointegrin a₇b₁ on skeletal muscle cells. Galectin 3 has also beenobserved to bind specifically to laminin, immunoglobulin E and itsreceptor, and bacterial lipopolysaccharides.

Various galectins have been shown to function in the mechanisms ofintercellular communication. For example, depending on cell type,galectin 1 has been observed to modulate cell adhesion either positivelyor negatively. More specifically, galectin 1 appears to inhibit celladhesion of skeletal muscle presumably by galectin 1-mediated disruptionof laminin-integrin a₇b₁ interactions. Alternatively, galectin 1 appearsto promote cell adhesion in several non-skeletal muscle cell typesexamined presumably by a glycoconjugate cross-linking mechanism.Galectin 3 has also been observed to function in modulatingcell-adhesion, as well as in the activation of certain immune cells bycross-linking IgE and IgE receptors. In addition, galectins have beenobserved to be involved in the regulation of immune cell activity, aswell as in such diverse processes as cell adhesion, proliferation,inflammation, autoimmunity, and metastasis of tumor cells. Furthermore,a galectin-like antigen designated HOM-HD-21 was recently found to behighly expressed in a Hodgkin's Disease cDNA library. Very recently, anovel galectin, termed PCTA-1, was identified as a specific cell surfacemarker on human prostate cancer cell lines and patient-derivedcarcinomas. Galectins have also been found to function intracellularlyas a component of ribonucleoprotein complexes. Finally, galectins 1 and3 have each been found to modulate T-cell growth and apoptosis byinteraction with CD45 and possibly Bcl2, respectively.

A relatively new family of cell-surface proteins has been identified andtermed the Ly6 superfamily. The members of this family include murineand human SCA-2, rat Ly-6 (also termed ThB), human CD59 [also known asprotectin or membrane attack complex inhibition factor (MACIF)], and E48antigen. The determination of an initial functional role for SCA-2 maylie in an analysis of its expression profile with regard to the complexprocess of hematopoiesis. SCA-2 is highly expressed in early thymicprecusor cells. In turn, progeny of the intrathymic precusor populationcontinue to express SCA-2, but only until the point of transition occursfrom blast cell to small cell. Further experimental evidencedemonstrates that mature thymocytes and peripheral T-cells do notexpress detectable levels of SCA-2, whereas mature, peripheral B-cellsdo continue to express SCA-2. As a result, it seems very likely thatSCA-2 plays an important role in thymocyte maturation anddifferentiation. A plausible explanation for this functional hypothesisis that SCA-2 may act as a receptor for a unknown cytokine whichregulates thymocyte maturation and differentiation.

In addition, CD59 is a recently identified integral membrane proteinwhich appears to be involved in the regulation of complement. Recentstudies show that the CD59 antigen may prevent damage from complementC5b-9 and protect astrocytes during inflammatory and infectiousdisorders of the nervous system. Expression of recombinant human CD59 onporcine donor organs have been shown to prevent complement-mediatedlysis and activation of endothelial cells that leads to hyperacuterejection. Recently, researchers at Alexion Pharmaceuticals (New Haven,Conn.) reported on the production of transgenic pigs which expressedhuman CD59. In these animals, xenogeneic organs were resistant tohyperacute rejection. (Fodor, et al., “Expression of a functional humancomplement inhibitor in a transgenic pig as a model for the preventionof xenogeneic hyperacute organ rejection,” Proc. Natl. Acad. Sci.,91:1153-11157 (1994).) The same company also reported that expression ofrecombinant transmembrane CD59 in paroxysmal nocturnal hemoglobinuria(PNH) B-cells confers resistance to human complement. (Rother et al.,“Expression of recombinant transmembrane CD59 in paroxysmal nocturnalhemoglobinuria B-cells confers resistance to human complement,” Blood,84:2604-2611 (1994).) PNH is an acquired hematopoietic disordercharacterized by complement-mediated hemolytic anemia, pancytopenia, andvenous thrombosis. It is thought that retroviral gene therapy with thismolecule could provide a treatment for PNH patients.

A final Ly6 superfamily member, the E48 antigen, is involved inintercellular adhesion between keratinocyte cells of the squamousepithelium. Such keratinocytes are attached to adjoining cells by largenumbers of desmosomes, which are thought to play a role in thetransition of transformed keratinocytes to metastatic tumor cells.Treatment with a monoclonal antibody raised against the E48 antigen hasbeen successful in the eradication of residual, postoperative squamouscell carcinoma cells of the upper aerodigestive tract in several in vivomodels and, to some degree, in humans. (van Dongen, et al., “Progress inradioimmunotherapy of head and neck cancer,” Oncol. Rep. 1:259-264(1994).) The gene encoding the E48 antigen has been mapped to theq24-qter region of human chromosome 8. Interestingly, a number of humandiseases have been mapped to this region of chromosome 8 includingLanger-Giedion syndrome, brachio-otorhinolaryngeal syndrome,trichorhinolaryngeal syndrome, and epidermolysis bullosa simplex.

A further example of a receptor family includes the prohibitinreceptors. The prohibitin gene product is expressed in a wide variety oftissues and has been implicated as a component of a number ofanti-proliferative mechanisms. The prohibitin gene encodes a 30 kDpostsynthetically modified polypeptide located primarily in themitochondria, but also may be associated with the IgM receptor on theB-cell plasma membrane. The protein functionally inhibits DNA synthesisand entry into S phase of the cell cycle by an unknown mechanism.Interestingly, although the prohibitin gene product is hypothesized tobe involved in the maintenance of senescence and the prevention ofcancer, one study found that, although somatic mutations in theprohibitin gene were present in a small number of breast cancers, nomutations were identified in any other breast, ovary, liver, and lungcancers examined. (Sato et al., Genomics 17:762-764 (1993).) However,the prohibitin gene has been mapped to human chromosome 17q12-21, thesame region thought to contain the gene involved in sporadic breastcancer. Furthermore, DNA sequence analysis of the prohibitin geneidentified somatic mutation in 4 of 23 cases of sporadic breast cancerexamined. Thus, prohibitin family members may be involved in thedevelopment of cancer.

Moreover, the EGFR family of plasma membrane proteins are an integralcomponent of normal cellular proliferation and in the pathogenesis ofthe cancerous state. The family is relatively small and includes theEGFR, c-erbB-2, c-erbB-3, and others. Various cancers are correlatedwith aberrant expression of one or more of these genes. A number ofligands have been identified which bind to the EGFR-like receptorslisted above including TGF-a, heparin-binding EGF, amphiregulin,criptoregulin, hercgulin, and others. A large fraction ofadenocarcinomas examined to date, especially those of the breast, colon,and pancreas, are typified by the amplification or overexpression of thec-erbB-2 gene. EGF, or an analogous ligand, initiates the cellulargrowth factor response by binding to the EGFR, or EGFR-related,receptor. Following the binding event, the receptor molecule dimerizesactivating its intracellular tyrosine kinase domain. This event resultsin the phosphorylation of specific tyrosine residues near the carboxyterminus of the receptor. The diversity of signals able to be transducedthrough the relatively small number of EGFR-related receptor moleculesis amplified considerably by the recent finding that EGFR-like receptormolecules can function when dimerized with other EGFR family membersforming heterodimers.

Members of the EGFR-related family of integral membrane proteins havebeen implicated in the pathogenesis of a number of human disease-states.For example, a mutation in the EGFR itself appears to play an importantrole in the development of glioblastomas. (Sang et al., J. Neurosurg82:841-846 (1995).) The EGFR gene is amplified or overexpressed in themajority of primary human glioblastomas. Although not conferring adistinct advantage on cell growth, an increase in EGFR expression wasfound to confer an increase in the ability of glioma cells to maintainanchorage-independent growth in soft agar especially in response to EGFand retinoic acid. Anchorage-independent growth in vitro correlateshighly with tumorigenicity in vivo, therefore, it is likely that cellswhich express abnormally high levels of EGFR in human glioblastoma cellsmay be involved in the high potential for these cells to cause tumors invivo.

Moreover, overexpression or amplification of c-erbB-2 has been reportedto be involved in a high number adenocarcinomas, particularly of thebreast, colon, and pancreas, and in a small proportion of ovariancarcinomas.

Thus, there is a clear need for identifying and exploiting novel membersof the receptor families, such as those described above. Althoughstructurally related, these receptors will likely possess diverse andmultifaceted functions in a variety of cell and tissue types. Receptortype molecules should prove useful in target based screens for smallmolecules and other such pharmacologically valuable factors. Monoclonalantibodies raised against such receptors may prove useful astherapeutics in an anti-tumor, diagnostic, or other capacity.Furthermore, receptors described here may prove useful in an active orpassive immunotherapeutical role in patients with cancer or otherimmunocompromised disease states.

BRIEF SUMMARY OF THE INVENTION

This invention relates to newly identified polynucleotides and thepolypeptides encoded by them, the use of such polynucleotides andpolypeptides, and their production. More particularly, thepolynucleotides and polypeptides of the present invention relate tospecific receptor families described in the specification and known inthe art. The invention also relates to inhibiting or activating theaction of such polynucleotides and polypeptides.

In one aspect, the invention relates to receptor polypeptides andpolynucleotides, as well as the methods for their production. Anotheraspect of the invention relates to methods for using such receptorpolypeptides and polynucleotides. Such uses include the treatment of thespecified diseases, among others. In still another aspect, the inventionrelates to methods to identify agonists and antagonists using thematerials provided by the invention, and treating conditions associatedwith receptor imbalance with the identified compounds. Yet anotheraspect of the invention relates to diagnostic assays for detectingdiseases associated with inappropriate receptor activity or levels.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B shows an amino acid sequence alignment of Clone ID HMACR70(SEQ ID NO:18) versus OB-1 (SEQ ID NO:33) (shaded boxes indicateidentical amino acid residues, non-shaded boxes indicate conservativesubstitutions).

FIG. 2 shows an amino acid sequence alignment of Clone ID HTEDK48 (SEQID NO:19) versus MRC-OX44 (SEQ ID NO:34) and PETA-3 (SEQ ID NO:35)(shaded boxes indicate identical amino acid residues, non-shaded boxesindicate conservative substitutions).

FIG. 3 shows an amino acid sequence alignment of Clone ID HPWAE25 (SEQID NO:20) versus NAG-2 (SEQ ID NO:36) and TALLA-1 (SEQ ID NO:37) (shadedboxes indicate identical amino acid residues, non-shaded boxes indicateconservative substitutions).

FIG. 4 shows an amino acid sequence alignment of Clone ID HTPEF86 (SEQID NO:21) versus B1 (SEQ ID NO:38) (shaded boxes indicate identicalamino acid residues, non-shaded boxes indicate conservativesubstitutions).

FIG. 5 shows an amino acid sequence alignment of Clone ID HSBBF02 (SEQID NO:22) versus TALLA-1 (SEQ ID NO:37) (shaded boxes indicate identicalamino acid residues, non-shaded boxes indicate conservativesubstitutions).

FIG. 6 shows an amino acid sequence alignment of Clone ID HLTAH80 (SEQID NO:23) versus TALLA-1 (SEQ ID NO:37) (shaded boxes indicate identicalamino acid residues, non-shaded boxes indicate conservativesubstitutions).

FIG. 7 shows an amino acid sequence alignment of Clone ID HTPBA27 (SEQID NO:24) versus NAG-2 (SEQ ID NO:36)(shaded boxes indicate identicalamino acid residues, non-shaded boxes indicate conservativesubstitutions).

FIG. 8 shows an amino acid sequence alignment of Clone ID HAIDQ59 (SEQID NO:25) versus CD9 (SEQ ID NO:39) (shaded boxes indicate identicalamino acid residues, non-shaded boxes indicate conservativesubstitutions).

FIG. 9 shows an amino acid sequence alignment of Clone ID HHFEK40 (SEQID NO:26) versus PETA-3 (SEQ ID NO:35) (shaded boxes indicate identicalamino acid residues, non-shaded boxes indicate conservativesubstitutions).

FIG. 10 shows an amino acid sequence alignment of Clone ID HGBGV89 (SEQID NO:27) versus L6H (SEQ ID NO:40) (shaded boxes indicate identicalamino acid residues, non-shaded boxes indicate conservativesubstitutions).

FIG. 11 shows an amino acid sequence alignment of Clone ID HUVBB80 (SEQID NO:28) versus L6 (SEQ ID NO:41) (shaded boxes indicate identicalamino acid residues, non-shaded boxes indicate conservativesubstitutions).

FIG. 12 shows an amino acid sequence alignment of Clone ID HJACE54 (SEQID NO:29) versus rGALECTIN-5 (SEQ ID NO:42) and hGALECTN-8 (SEQ IDNO:43) (shaded boxes indicate identical amino acid residues, non-shadedboxes indicate conservative substitutions).

FIG. 13 shows an amino acid sequence alignment of Clone ID HROAD63 (SEQID NO:30) versus E48 (SEQ ID NO:44) (shaded boxes indicate identicalamino acid residues, non-shaded boxes indicate conservativesubstitutions).

FIG. 14 shows an amino acid sequence alignment of Clone ID HMWGS46 (SEQID NO:31) versus B-cell Receptor Associated Protein (shaded boxesindicate identical amino acid residues, non-shaded boxes indicateconservative substitutions).

FIG. 15 shows an amino acid sequence alignment of Clone ID HNFGW06 (SEQID NO:32) versus EGFR (SEQ ID NO:46) (shaded boxes indicate identicalamino acid residues, non-shaded boxes indicate conservativesubstitutions).

DETAILED DESCRIPTION OF THE INVENTION Definitions

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“Receptor” refers, among others, to a polypeptide comprising the aminoacid sequence set forth in SEQ ID NO:Y, or an allelic variant thereof.

“Receptor Activity” or “Biological Activity of the Receptor” refers tothe metabolic or physiologic function of said receptor including similaractivities or improved activities or these activities with decreasedundesirable side-effects. Also included are antigenic and immunogenicactivities of said receptor.

“Receptor gene” refers to a polynucleotide comprising the nucleotidesequence set forth in SEQ ID NO:X or allelic variants thereof and/ortheir complements.

“SEQ ID NO:X” comprises all or a substantial portion of thepolynucleotide encoding each receptor of the invention. The value X forthe nucleotide sequence is an integer specified in Table 1. Thisnucleotide sequence was translated into the receptor polypeptideidentified in Table 1 as “SEQ ID NO:Y,” where the value of Y for eachreceptor polypeptide is an integer defined in Table 1.

The invention further provides a composition of matter comprising anucleic acid molecule which comprises a human cDNA clone identified by acDNA Clone ID (Identifier) in Table 1, which DNA molecule is containedin the material deposited with the American Type Culture Collection(“ATCC™”) and given the ATCC™ Deposit Number shown in Table 1 for thatcDNA clone. The ATCC™ is located at American Type Culture Collection(ATCC™), 10801 University Boulevard, Manassas, Va. 20110-2209, USA. Thedeposit has been made under the terms of the Budapest Treaty on theinternational recognition of the deposit of micro-organisms for purposesof patent procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. The deposit is provided merely as convenience to those of skillin the art and is not an admission that a deposit is required forenablement, such as that required under 35 U.S.C. §112. The nucleotidesequence of the polynucleotides contained in the deposited material, aswell as the amino acid sequence of the polypeptide encoded thereby, arecontrolling in the event of any conflict with any description ofsequences herein.

“Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immunoglobulinexpression library.

“Isolated” means altered “by the hand of man” from the natural state. Ifan “isolated” composition or substance occurs in nature, it has beenchanged or removed from its original environment, or both. For example,a polynucleotide or a polypeptide naturally present in a living animalis not “isolated,” but the same polynucleotide or polypeptide separatedfrom the coexisting materials of its natural state is “isolated”, as theterm is employed herein.

“Polynucleotide” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications has been made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

“Polypeptide” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. “Polypeptide” refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids.“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from posttranslation natural processes ormay be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. (See, for instance, PROTEINS—STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W.H. Freeman and Company, NewYork, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al., “Analysis for protein modifications and nonproteincofactors”, Meth Enzymol (1990) 182:626-646 and Rattan et al., “ProteinSynthesis: Posttranslational Modifications and Aging”, Ann NY Acad Sci(1992) 663:48-62.)

“Variant” as the term is used herein, is a polynucleotide or polypeptidethat differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniquesor by direct synthesis.

“Identity” is a measure of the identity of nucleotide sequences or aminoacid sequences. In general, the sequences are aligned so that thehighest order match is obtained. “Identity” per se has an art-recognizedmeaning and can be calculated using published techniques. (See, e.g.:COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS,Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OFSEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, vonHeinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.)While there exist a number of methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans. (Carillo, H., and Lipton, D., SIAM J AppliedMath (1988) 48:1073.) Methods commonly employed to determine identity orsimilarity between two sequences include, but are not limited to, thosedisclosed in Guide to Huge Computers, Martin J. Bishop, ed., AcademicPress, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J AppliedMath (1988) 48:1073. Methods to determine identity and similarity arecodified in computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to, GCS program package (Devereux, J., et al., Nucleic AcidsResearch (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. etal., J Molec Biol (1990) 215:403.)

As an illustration, by a polynucleotide having a nucleotide sequencehaving at least, for example, 95% “identity” to a reference nucleotidesequence of SEQ ID NO:X is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence of SEQ ID NO: X. Inother words, to obtain a polynucleotide having a nucleotide sequence atleast 95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These mutations of the reference sequence may occur at the 5or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among nucleotides in the reference sequence or in one ormore contiguous groups within the reference sequence.

Similarly, by a polypeptide having an amino acid sequence having atleast, for example, 95% “identity” to a reference amino acid sequence ofSEQ ID NO:Y is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of SEQ ID NO:Y. In other words,to obtain a polypeptide having an amino acid sequence at least 95%identical to a reference amino acid sequence, up to 5% of the amino acidresidues in the reference sequence may be deleted or substituted withanother amino acid, or a number of amino acids up to 5% of the totalamino acid residues in the reference sequence may be inserted into thereference sequence. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

Polypeptides of the Invention

In one aspect, the present invention relates to receptor polypeptides(or receptor proteins). The receptor polypeptides include thepolypeptide of SEQ ID NO:Y; as well as polypeptides comprising the aminoacid sequence of SEQ ID NO:Y; and polypeptides comprising the amino acidsequence which have at least 80% identity to that of SEQ ID NO:Y overits entire length, and still more preferably at least 90% identity, andeven still more preferably at least 95% identity to SEQ ID NO:Y.Furthermore, those with at least 97-99% identity to SEQ ID NO:Y arehighly preferred. Also included within receptor polypeptides arepolypeptides having the amino acid sequence which have at least 80%identity to the polypeptide having the amino acid sequence of SEQ IDNO:Y over its entire length, and still more preferably at least 90%identity, and even still more preferably at least 95% identity to SEQ IDNO:Y. Furthermore, those with at least 97-99% are highly preferred.Preferably receptor polypeptides exhibit at least one biologicalactivity of the receptor.

The receptor polypeptides may be in the form of the “mature” protein ormay be a part of a larger protein such as a fusion protein. It is oftenadvantageous to include an additional amino acid sequence which containssecretory or leader sequences, pro-sequences, sequences which aid inpurification such as multiple histidine residues, or an additionalsequence for stability during recombinant production.

Fragments of the receptor polypeptides are also included in theinvention. A “fragment” is a polypeptide having an amino acid sequencethat entirely is the same as part, but not all, of the amino acidsequence of the aforementioned receptor polypeptides. As with receptorpolypeptides, fragments may be “free-standing,” or comprised within alarger polypeptide of which they form a part or region, most preferablyas a single continuous region. Representative examples of polypeptidefragments of the invention, include, for example, fragments from aboutamino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the endof receptor polypeptide. In this context “about” includes theparticularly recited ranges larger or smaller by several, 5, 4, 3, 2 or1 amino acid at either extreme or at both extremes.

Preferred fragments include, for example, truncation polypeptides havingthe amino acid sequence of receptor polypeptides, except for deletion ofa continuous series of residues that includes the amino terminus, or acontinuous series of residues that includes the carboxyl terminus ordeletion of two continuous series of residues, one including the aminoterminus and one including the carboxyl terminus.

Also preferred are fragments characterized by structural or functionaldomains, such as fragments that comprise alpha-helix and alpha-helixforming regions, beta-sheet and beta-sheet-forming regions, turn andturn-forming regions, coil and coil-forming regions, hydrophilicregions, hydrophobic regions, alpha amphipathic regions, betaamphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions. The“domains” of each receptor polypeptide are illustrated in the Figures.The Figures compare SEQ ID NO:Y to the closest know homologue. Identicalamino acids shared between the two polypeptides are shaded, whileconservative amino acid changes are boxed. By examining the regions oramino acids shaded and/or boxed, the skilled artisan can readilyidentify conserved domains between the two polypeptides. The amino acidssequences of SEQ ID NO:Y falling within these conserved domains are“fragments” and are specifically contemplated by the present invention.Especially preferred is the extracellular domains of a receptor of theinvention. Soluble extracellular domains have antagonist activitymediated by competition with a receptor ligand.

Other preferred fragments are biologically active fragments.Biologically active fragments are those that mediate receptor activity,including those with a similar activity or an improved activity, or witha decreased undesirable activity. Also included are those that areantigenic or immunogenic in an animal, especially in a human.

Preferably, all of these polypeptide fragments retain a biologicalactivity of the receptor, including antigenic activity. Variants of thedefined sequence and fragments also form part of the present invention.Preferred variants are those that vary from the referents byconservative amino acid substitutions—i.e., those that substitute aresidue with another of like characteristics. Typical such substitutionsare among Ala, Val, Leu and Ile; among Ser and Thr; among the acidicresidues Asp and Glu; among Asn and Gln; and among the basic residuesLys and Arg; or aromatic residues Phe and Tyr. Particularly preferredare variants in which several, 5-10, 1-5, or 1-2 amino acids aresubstituted, deleted, or added in any combination.

The receptor polypeptides of the invention can be prepared in anysuitable manner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

Polynucleotides of the Invention

Another aspect of the invention relates to receptor polynucleotides.Receptor polynucleotides include isolated polynucleotides which encodethe receptor polypeptides and fragments, and polynucleotides closelyrelated thereto. More specifically, a receptor polynucleotide of theinvention includes a polynucleotide comprising the nucleotide sequencecontained in SEQ ID NO:X encoding a receptor polypeptide of SEQ ID NO:Y,and polynucleotide having the particular sequence of SEQ ID NO:X.

Receptor polynucleotides further include a polynucleotide comprising anucleotide sequence that has at least 80% identity over its entirelength to a nucleotide sequence encoding the receptor polypeptide of SEQID NO:Y, and a polynucleotide comprising a nucleotide sequence that isat least 80% identical to that of SEQ ID NO:X over its entire length. Inthis regard, polynucleotides at least 90% identical are particularlypreferred, and those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred and those withat least 98-99% are most highly preferred, with at least 99% being themost preferred. Also included under receptor polynucleotides are anucleotide sequence which has sufficient identity to a nucleotidesequence contained in SEQ ID NO:X, or contained in the cDNA insert inthe plasmid deposited with ATCC™, to hybridize under conditions useablefor amplification or for use as a probe or marker. Moreover, thereceptor polynucleotide includes a nucleotide sequence having at least80% identity to a nucleotide sequence encoding the receptor polypeptideexpressed by the cDNA insert deposited at the ATCC™, and a nucleotidesequence comprising at least 15 contiguous nucleotides of such cDNAinsert. In this regard, polynucleotides at least 90% identical areparticularly preferred, and those with at least 95% are especiallypreferred. Furthermore, those with at least 97% are highly preferred andthose with at least 98-99% are most highly preferred, with at least 99%being the most preferred. The invention also provides polynucleotideswhich are complementary to all the above receptor polynucleotides.

The receptors of the invention are structurally related to otherproteins of specified receptor families, as shown by the results in theFigures. The cDNA sequence of SEQ ID NO:X encodes a polypeptide asdescribed in Table 1 as SEQ ID NO:Y. Because the receptor polypeptidescontain domains similar in structure to other receptor family members,the receptors of the present invention are expected to have, inter alia,similar biological functions/properties to their homologous polypeptidesand polynucleotides, and their utility is obvious to anyone skilled inthe art.

TABLE 1 SEQ ID SEQ ID ATCC ™ ATCC ™ Receptor Clone ID Name NO: X NO: YDeposit No. Deposit Date Family Homology HMACR70 1 18 209054 May 16,1997 Ig Sialoadhesin ##### Jan. 21, 1998 OB-1 HTEDK48 209054 May 16,1997 TM4SF MRC-OX44 PETA-3 1-1849 bp 2 160-900 bp 3 19 HTPED39 4 20209054 May 16, 1997 TM4SF NAG-2 HPWAE25 ##### Jan. 21, 1998 TALLA-1HTPEF86 5 21 209053 May 16, 1997 TM4SF CD20 B1 Antigen HSBBF02 6 22209054 May 16, 1997 TM4SF TALLA-1 HLTAH80 7 23 97242 Aug. 02, 1995 TM4SFTALLA-1 209054 May 16, 1997 HTPBA27 8 24 97242 Aug. 02, 1995 TM4SF NAG-2209054 May 16, 1997 HAIDQ59 209054 May 16, 1997 TM4SF CD9 Antigen 5′Sequence 9 25 3′ Sequence 10 - HHFEK40 11 26 209054 May 16, 1997 TM4SFPETA-3 HGBGV89 12 27 209125 Jun. 09, 1997 TM4SF L6H 209054 May 16, 1997HUVBB80 13 28 209054 May 16, 1997 TM4SF L6 HJACE54 14 29 209053 May 16,1997 Lectin Galectin-3 Galectin-5 Galectin-8 HROAD63 15 30 209053 May16, 1997 Ly6 E48 splice variant HMWGS46 16 31 209053 May 16, 1997Prohibitin BAP-37 HNFGW06 17 32 209053 May 16, 1997 EGFR EGFR

The novel full-length cDNA clone designated HMACR70 (SEQ ID NO:1) may bea member of the sialoadhesin family of the Ig superfamily ofreceptor-like molecules and a CD33 homologue. HMACR70 contains a 1497nucleotide cDNA insert (SEQ ID NO:1) encoding a 315 amino acid ORF (SEQID NO:18) and was cloned from a GM-CSF-treated human macrophage cDNAlibrary. The only additional cDNA libraries in the HGS database whichinclude this clone are human eosinophils and possibly human gallbladder. A BLAST analysis of the amino acid sequence of HMACR70 (SEQ IDNO:18) demonstrates that this clone exhibits approximately 50% identityand 69% similarity over a 300 amino acids stretch of a gene termed humandifferentiation antigen, and 38% identity and 62% similarity of thehuman myelin-associated glycoprotein precursor CD33 gene.

A more recent BLAST analysis confirms HMACR70's (SEQ ID NO:18)designation as a sialoadhesin family member. HMACR70 (SEQ ID NO:18) ishomologous to two recently identified sialoadhesin family members, humanOB binding protein (OB) 1 (SEQ ID NO:33) and 2. (See, Genbank AccessionNo. U71382; see FIG. 1.) It is thought that OB-1 (SEQ ID NO:33) and OB-2may bind leptin. Thus, HMACR70 (SEQ ID NO:18), as a sialoadhesin familymember, may act to attenuate or even amplify intercellular routes ofcommunication, including binding to leptin or modulating the activity ofimmune cells, such as macrophages. Clearly, any diseases affected bythese processes could be treated by the polypeptide or fragment ofHMACR70 (SEQ ID NO:18).

The full-length nucleotide sequences of ten novel human cDNA cloneswhich potentially belong to the TM4SF superfamily are disclosed in thetable above and will be addressed sequentially.

The cDNA clone HTEDK48 contains a 1849 nucleotide cDNA insert (SEQ IDNO:2) encoding a 245 amino acid ORF that was cloned from a human testescDNA library. The coding sequence of HTEDK48 (SEQ ID NO: 3) may be fusedto other human proteins, such as 3-hydroxyacyl-CoA dehydrogenase. BLASTanalysis of the amino acid sequence of HTEDK48 (SEQ ID NO:19)demonstrates that this clone exhibits approximately 30% identity and 51%similarity over a 245 amino acid stretch of the CD82 molecule. Recentstudies have shown that CD82 can associate with CD4 or CD8 and delivercostimulatory signals for the TCR/CD3 pathway. CD82 has also been foundto be involved in syncytium formation in HTLV-1-infected T-cells. Andfinally, in a recently published study in which the expression of theCD82 gene by tumors of the lung was examined retrospectively, it wasreported that CD82 may be linked to the suppression of tumor metastasisof prostate cancer. The study also reported that decreased CD82expression may be involved in malignant progression of such cancers.Thus, HTEDK48 (SEQ ID NO:2, NO:3, NO:19) may also be involved in thedevelopment of cancer.

A more recent BLAST analysis shows that HTEDK48 (SEQ ID NO:19) ishomologous the rat leukocyte antigen, MRC OX-44 (SEQ ID NO:34), and theplatelet endothelial tetraspan antigen-3 (PETA-3) (SEQ ID NO:35). (SeeFIG. 2X.) MRC OX-44 (SEQ ID NO:34), a member of a new family of cellsurface proteins, appears to be involved in growth regulation. (See,Bellacosa, A., et al., “The Rat Leukocyte antigen MRC OX-44 is a Memberof a New Family of Cell Surface Proteins which Appear to be Involved inGrowth Regulation,” Mol. Cell. Bio. 11: 2864-2872 (1991).) Similarly,PETA-3 (SEQ ID NO:35) has been located to platelet endothelial cells,and an anti-PETA-3 antigen monoclonal antibody can stimulate plateletaggregation and mediator release. (See, Fitter, S., “Molecular Cloningof cDNA Encoding a Novel Platelet-Endothelial Cell Tetra-Span Antigen,PETA-3,” Blood, 86(4):1348-1355 (1995).) Thus, HTEDK48 (SEQ ID NO:19)may function similar to MRC OX-44 (SEQ ID NO:34) or PETA-3 (SEQ IDNO:35) to affect growth of blood cells. Administering polypeptides orfragments of HTEDK48 (SEQ ID NO:19) may be an effective treatment ofblood disorders.

The cDNA clone HPWAE25 contains a 1288 nucleotide cDNA insert (SEQ IDNO:4) encoding a 273 amino acid ORF (SEQ ID NO:20) that was cloned froma human pancreas tumor cDNA library, while clone HTPED39 represents atruncated cDNA sequence. This clone also appears in a number of othercDNA libraries constructed from a variety of human cell and tissue typesincluding keratinocytes, ulcerative colitis, striatum depression, lymphnode breast cancer, ovarian cancer, stage B2 prostate cancer, kidneymedulla, and others. Northern blot analysis of HLTAH80 (SEQ ID NO:23)also shows expression in a variety of human cell lines including U937,MM96, WM115, and MDAMB231. A BLAST analysis of the amino acid sequenceof HTPED39 demonstrates that this clone exhibits approximately 35%identity and 50% similarity over the entire length of the CD37 molecule.The CD37 antigen is expressed on B cells and on a subpopulation of Tcells, but not on pre-B or plasma cells. It has been reported that CD37expression is downregulated in conjunction with B-cell activation,suggesting that CD37 may be involved in the processes which dictate theactivation state of the B-cell.

Moreover, HPWAE25 (SEQ ID NO:20) is also homologous to recentlyidentified TM4SF members, NAG-2 (SEQ ID NO:36) and TALLA-1 (SEQ IDNO:37). (See FIG. 3.) NAG-2 (SEQ ID NO:36) is thought to complex withintegrins and other TM4SF proteins, while TALLA-1 (SEQ ID NO:37) is ahighly specific marker of T-cell acute lymphoblastic leukemia andneuroblastoma. (See, Tachibana, I., et al., “NAG-2, A NovelTransmembrane-4 Superfamily (TM4SF) Protein that Complexs with Integrinsand Other TM4SF Proteins,” J. Biol. Chem., 272:29181-29189 (1997);Takagi, S., “Identification of a Highly Specific Surface Marker ofT-cell Acute Lymphoblastic Leukemia and Neuroblastoma as a New Member ofthe Transmembrane 4 Superfamily,” Int. J. Cancer 61(5):706-715 (1995).)Thus, HPWAE25 (SEQ ID NO:20) may be involved the development of cancer,particularly leukemia, lymphoma, and neuroblastoma. HPWAE25 (SEQ ID NO:4and NO:20) may be used as an effective treatment of these cancers, aswell as a diagnostic marker.

A subfamily of TM4SF receptors include CD20 proteins. A CD20-like cDNAclone was obtained from a human pancreas tumor cDNA library and containsa 1236 nucleotide insert which encodes a 250 amino acid ORF. A BLASTanalysis of the deduced amino acid sequence of HTPEF86 (SEQ ID NO:21)exhibits approximately 41% identity and 61% similarity to the CD20 gene,also known as B1 antigen (SEQ ID NO:38). (See FIG. 4.) Expression ofthis gene is detected in only two additional HGS human cDNA libraries;amygdala depression and 9 week early stage human. Although the precisefunctional role of CD20 has yet to be determined, it is clear that CD20plays a key role in the regulation of B-cell activation. Based primarilyon sequence identity, the novel CD20-like molecule presented herein mayalso be involved in cell cycle activation. Potential therapeutic and/ordiagnostic applications for HTPEF86 (SEQ ID NO:21) may include suchclinical presentations as juvenile rheumatoid arthritis, Graves'Disease, and a number of B-cell lymphomas or other lymphoid tumors.

The clone HSBBF02 contains a 1115 nucleotide cDNA insert (SEQ ID NO:6)encoding a 245 amino acid ORF (SEQ ID NO:22) and was cloned from an HSC172 cell line cDNA library. This clone also appears in a number of othercDNA libraries constructed from a variety of human cell and tissue typesincluding brain amygdala depression, endothelial cells, fetal liver andheart, osteoblasts, testes, and others. A BLAST analysis of the aminoacid sequence of HSBBF02 (SEQ ID NO:22) demonstrates that this cloneexhibits approximately 64% identity and 80% similarity with the A15molecule over a 131 amino acid stretch (A15 is composed of 244 aminoacids). A more recent BLAST search shows that HSBBF02 (SEQ ID NO:22) issimilar to the TALLA-1 protein (SEQ ID NO:37) and may in fact be aclosely related family member. (See FIG. 5.)

In addition, a second cDNA clone, designated HLTAH80 (SEQ ID NO:23),exhibits sequence similarity to the A15 molecule and TALLA-1 (SEQ IDNO:37). (See FIG. 6.) This clone contains a 1662 nucleotide cDNA insertencoding a 253 amino acid ORF and was cloned from a human T-celllymphoma cDNA library. This clone also appears in a number of other cDNAlibraries constructed from a variety of human cell and tissue typesincluding B-cell lymphoma, corpus collosum, endometrial tumor,osteosarcoma, testes, and others. Northern blot analysis of HLTAH80 (SEQID NO:7) also shows expression in a variety of human tissues includingspleen, lymph node, thymus, PBLs, heart, and a particularly strongsignal in skeletal muscle and pancreas. A BLAST analysis of the aminoacid sequence of HLTAH80 (SEQ ID NO:23) demonstrates that this cloneexhibits approximately 35% identity and 55% similarity over the entirelength of the A15 molecule.

Since expression of A15 drops to undetectable levels when comparingimmature T-cells to peripheral blood lymphocytes, it is thought that A15may play a role in the development of T-cells. Furthermore, theMXS1(CCG-B7) gene which codes for A15 contains a number of tripletnucleotide repeats which have been associated with neuropsychiatricdiseases such as Huntington's chorea, fragile X syndrome, and myotonicdystrophy. In addition, A15 appears to be expressed exclusively onT-cell acute lymphoblastic leukemia cell lines, including severalderived from adult T-cell leukemia and those established byimmortalization with human T-cell leukemia virus type 1 or Herpesvirussaimiri. Thus, clones HLTAH80 (SEQ ID NO:7 and NO:23) and/or HSBBF02(SEQ ID NO:6 and NO:22) may also be involved in diseases caused by theexpansion of repeats or chromosomal instability.

The cDNA clone HTPBA27 contains a 1345 nucleotide cDNA insert (SEQ IDNO:8) encoding a 238 amino acid ORF (SEQ ID NO:24) and was cloned from ahuman tumor pancreas cDNA library. This clone also appears in a numberof other cDNA libraries constructed from a variety of human cell andtissue types including cerebellum, breast lymph node, osteosarcoma,adult testes, RS4; 11 bone marrow cell line, microvascular endothelialcells, and others. A BLAST analysis of the amino acid sequence ofHTPBA27 (SEQ ID NO:24) demonstrates that this clone exhibitsapproximately 40% identity and 64% similarity with a glycoprotein termedCD53 over its entire length. CD53 is thought to be involved inthymopoiesis, since rat CD53 can be detected on immatureCD4-8-thymocytes and the functionally mature single-positive subset, butcannot be detected on the intermediate CD4+8+ thymocytic subset ofcells. The CD53 molecule has also been implicated as a component ofsignal transduction pathways in B cells, monocytes and granulocytes, ratmacrophages, NK, and T cells. Moreover, as illustrated in FIG. 7,HTPBA27 (SEQ ID NO:24) was recently confirmed as a TM4SF receptor. (See,Tachibana, I., et al., “NAG-2, A Novel Transmembrane-4 Superfamily(TM4SF) Protein that with Integrins and Other TM4SF Proteins,” J. Biol.Chem., 272:29181-29189 (1997).) Calling the HTPBA27 polypeptide (SEQ IDNO:24) NAG-2 (SEQ ID NO:36), this group confirmed HTPBA27's status as aTM4SF receptor by showing that NAG-2 (SEQ ID NO:36) complexes withintegrin and other TM4SF receptors. Thus, diseases caused by the failureof HTPBA27 (SEQ ID NO:24) to complex with integrin and other TM4SFreceptors can be treated by administering HTPBA27(SEQ ID NO:24). HTPBA27(SEQ ID NO:8 and NO:24) can also be used to diagnose these diseases.

The cDNA clone HAIDQ59 contains cDNA insert encoding a 221 amino acidORF (SEQ ID NO:25) that was cloned from a human epithelial cell inducedwith TNFa and INF cDNA library. The 5′ end of HAIDQ59 is represented bythe SEQ ID NO: 9, while the 3′ end is represented by SEQ ID NO: 10. Thisclone appears in only two additional cDNA libraries in the HGS database.These two libraries were constructed from the human Jurkat T-cell lineand human microvascular endothelial cells. A BLAST analysis of the aminoacid sequence of HAIDQ59 (SEQ ID NO:25) demonstrates that this cloneexhibits approximately 53% identity and 69% similarity over 226 aminoacids of the CD9 TM4SF molecule (SEQ ID NO:39). (See FIG. 8.) It hasbeen demonstrated that the CD9 molecule (SEQ ID NO:39) is involved insignal transduction pathways in platelets, as well as in cell adhesionin both platelets and pre-B-cell lines. Intriguingly, a monoclonalantibody (vpg15), which recognizes the feline homologue of CD9, has beenshown to block infection by feline immunodeficiency virus (FIV).Furthermore, a recent study shows that cells expressing high levels ofCD9 (SEQ ID NO:39) exhibited suppressed cell motility. Thus, HAIDQ59(SEQ ID NO:25) may also be involved in signal transduction of bloodcells.

The cDNA clone HHFEK40 contains a 936 nucleotide cDNA insert (SEQ IDNO:11) encoding a 252 amino acid ORE (SEQ ID NO:26) and was cloned froma human fetal heart cDNA library. This clone appears once in the humanfetal heart cDNA library and possibly in a hemangiopericytoma cDNAlibrary. A BLAST analysis of the amino acid sequence of HHFEK40 (SEQ IDNO:26) demonstrated that this clone exhibits approximately 60% identityand 75% similarity over the entire length of a molecule designatedPETA-3 (SEQ ID NO:35). (See FIG. 9.) PETA-3 (SEQ ID NO:35) wasoriginally identified as a novel human platelet surface glycoproteintermed gp27. Although PETA-3 (SEQ ID NO:35) is present in low abundanceon the platelet surface, an anti-PETA-3 monoclonal antibody canstimulate platelet aggregation and mediator release. Thus, HHFEK40 (SEQID NO:26) may function similar to PETA-3 (SEQ ID NO:35) to affect growthof blood cells. Administering polypeptides or fragments of HHFEK40 (SEQID NO:26) may be an effective treatment of blood disorders.

The cDNA clone HGBGV89 contains a 738 nucleotide cDNA insert (SEQ IDNO:12) encoding a 197 amino acid ORF (SEQ ID NO:27) and was cloned froma human gall bladder cDNA library. The only two additional appearancesof this clone in the HGS database are in a normalized fetal liver cDNAlibrary and in a fetal liver/spleen cDNA library. The cDNA clone HUVBB80contains a 1071 nucleotide cDNA insert (SEQ ID NO:13) encoding a 201amino acid ORF (SEQ ID NO:28) and was cloned from a human umbilical veincDNA library. This clone appears in several additional cDNA libraries inthe HGS database including prostate BPH, thyroid, and fetalliver/spleen. BLAST analyses of the amino acid sequences of HGBGV89 (SEQID NO:27) and HUVBB80 (SEQ ID NO:28) demonstrate that these clonesexhibit approximately 49% identity and 65% similarity and 47% identityand 68% similarity, respectively, over the entire length of a moleculedesignated L6 surface protein (SEQ ID NO:41) or human tumor-associatedantigen L6 (SEQ ID NO:41) (See FIGS. 10 & 11.) Moreover, another grouphas confirmed the TM4SF receptor homology of HGBGV89 (SEQ ID NO:27) bydescribing the protein as a putative transmembrane protein L6H (SEQ IDNO:40). (See Genbank Accession No 2587054; see FIG. 10.) The L6 cellsurface antigen (SEQ ID NO:41) is highly expressed on lung, breast,colon, and ovarian carcinomas. Promising results of phase 1 clinicalstudies have been reported with an anti-L6 monoclonal antibody, or itshumanized counterpart, suggesting that the L6 antigen (SEQ ID NO:41) maybe an attractive target for monoclonal antibody-based cancer therapy.

In summary, there is a clear need for identifying and exploiting novelmembers of the TM4SF superfamily such as those described herein.Although structurally related, these factors will likely possess diverseand multifaceted functions in a variety of cell and tissue types.Receptor type molecules, such as the novel potential members of theTM4SF superfamily detailed here, should prove useful in target basedscreens for small molecules and other such pharmacologically valuablefactors. Monoclonal antibodies raised against such factors may proveuseful as therapeutics in an anti-tumor, diagnostic, or other capacity.Furthermore, factors such as the nine novel TM4SF superfamily-likemolecules described here may prove useful in an active or passiveimmunotherapeutical role in patients with cancer or otherimmunocompromised disease states.

Besides TM4SF receptors, receptors from other families are alsodescribed. For example, clone HJACE54 (SEQ ID NO:14 and NO:29), alsocalled galectin 11, exhibits significant sequence identity to the ratgalectin 5 (SEQ ID NO:42), the chicken galectin 3 gene, and the humangalectin 8 (SEQ ID NO:43) genes. (See FIG. 12.) The galectin 11 cDNAclone contains an 865 nucleotide insert (SEQ ID NO:14) which encodes a133 amino acid ORF (SEQ ID NO:29). The clone was obtained from a JurkatT-cell G1 phase cDNA library. A BLAST analysis of the deduced amino acidsequence of HJACE54 (SEQ ID NO:29) demonstrates approximately 35%identity and 57% similarity to the amino acid sequence of the ratgalectin 5 (SEQ ID NO:42) gene. Expression of galectin 11 (SEQ ID NO:14)is quite limited in the HGS database. In fact, the only two additionalESTs in the HGS database which contain the HJACE54 sequence (SEQ IDNO:14) were found in human neutrophil and human infant adrenal glandcDNA libraries. Northern blot analyses have not been performed toexamine expression patterns of the galectin 11 gene (SEQ ID NO:14).

Various galectins have been shown to function in the mechanisms ofintercellular communication. For example, depending on cell type,galectin 1 has been observed to modulate cell adhesion either positivelyor negatively. More specifically, galectin 1 appears to inhibit celladhesion of skeletal muscle presumably by galectin 1-mediated disruptionof laminin-integrin a₇b₁ interactions. Alternatively, galectin 1 appearsto promote cell adhesion in several non-skeletal muscle cell typesexamined presumably by a glycoconjugate cross-linking mechanism.Galectin 3 has also been observed to function in modulatingcell-adhesion, as well as in the activation of certain immune cells bycross-linking IgE and IgE receptors. In addition, galectins have beenobserved to be involved in the regulation of immune cell activity, aswell as in such diverse processes as cell adhesion, proliferation,inflammation, autoimmunity, and metastasis of tumor cells. Furthermore,a galectin-like antigen designated HOM-HD-21 was recently found to behighly expressed in a Hodgkin's Disease cDNA library. Very recently, anovel galectin, termed PCTA-1, was identified as a specific cell surfacemarker on human prostate cancer cell lines and patient-derivedcarcinomas. Galectins have also been found to function intracellularlyas a component of ribonucleoprotein complexes. Finally, galectins 1 and3 have each been found to modulate T-cell growth and apoptosis byinteraction with CD45 and possibly Bcl2, respectively. As a result, thediscovery of a novel galectin (SEQ ID NO:29), such as that encoded byHJACE54 (SEQ ID NO:14), is likely to be a valuable asset bothdiagnostically and therapeutically.

Additionally, a full-length nucleotide sequence of a novel human cDNAclone which encodes an apparent splice variant of the previouslydescribed human E48 antigen has recently been determined. (See FIG. 13.)Clone HROAD63 contains a 441 nucleotide cDNA (SEQ ID NO:15) whichencodes a 70 amino acid polypeptide (SEQ ID NO:30). This novel cloneexhibits significant sequence identity to several members of arelatively new family of cell-surface proteins termed the Ly6superfamily. These members include murine and human SCA-2, rat Ly-6(also termed ThB), and human CD59 [also known as protectin or membraneattack complex inhibition factor (MACIF)]. The novel E48 splice variant(SEQ ID NO:15) was obtained from the HGS human stomach cDNA library. Theclone (SEQ ID NO:30) is present in only a limited number of other HGScDNA libraries including kidney cancer, keratinocyte, and tongue. Analignment of the nucleotide sequences of the human E48 and HROAD63 (SEQID NO:15) cDNAs demonstrates that the initial 168 and 178 nucleotides ofE48 and HROAD63, respectively, are identical, with the exception of anadditional 10 nucleotides of sequence at the extreme 5′ end of theHROAD63 sequence. The sequence of the two clones is also identical foran additional 229 nucleotides including the 3′ end of the codingsequences and the entire 3′ untranslated regions. The only divergence ofnucleotide sequence in this region of the clones is the deletion of asingle thymidine residue in the 3′ UTR of the E48 cDNA. The majordifference between the two nucleotide sequences is a 329 nucleotidedeletion from the HROAD63 sequence. This deletion causes a shift in theHROAD63 reading frame and encompasses the translational stop signal usedin the E48 clone. As a result, the carboxy terminal sequence of HROAD63(SEQ ID NO:30) is radically altered with regard to that of E48 (SEQ IDNO:44) (as illustrated in FIG. 13 by the obvious differences betweenamino acids 56-128 of E48 and 56-70 of HROAD63 in the amino acidalignment). The clinical presentation of disorders, including abnormalskin and hair phenotypes, may be attributed, at least in part, to anon-functional Ly6 superfamily member such as E48 (SEQ ID NO:44) orHROAD63 (SEQ ID NO:30). HROAD63 (SEQ ID NO:30) may also be involved inblood disorders, as seen with its homologues SCA-2 and CD59.

A novel prohibitin cDNA clone (SEQ ID NO:16) presented herein wasoriginally identified in a human bone marrow cell line (RS4; 11) cDNAlibrary. The clone contains a 1066 nucleotide insert (SEQ ID NO:16)which encodes a 299 amino acid polypeptide (SEQ ID NO:31). BLAST andBestFit analyses of the predicted amino acid sequence of HMWGS46 (SEQ IDNO:31) demonstrate a highly significant sequence identity to a murineprotein termed IgM B-cell receptor associated protein (BAP)-37 (SEQ IDNO:45) (Genbank accession number X78683). The HMWGS46 amino acidsequence (SEQ ID NO:31) exhibits nearly perfect identity and similarityover the entire length of the murine BAP-37 sequence (SEQ ID NO:45).(See FIG. 14.) In addition, the full-length nucleotide sequences ofHMWGS46 (SEQ ID NO:16) and BAP-37 (SEQ ID NO:45) exhibit at least 87%identical. The HMWGS46 clone (SEQ ID NO:16) also exhibits approximately49% sequence identity and 85% sequence similarity to a human genedesignated prohibitin. Finally, the HMWGS46 cDNA (SEQ ID NO:16) appearsin a substantial number of HGS human cDNA libraries in addition to thebone marrow cell line cDNA library from which it was cloned. Some of thecDNA libraries in which this clone appears include keratinocytes,induced endothelial cells, activated neutrophils, synovial sarcoma,colon carcinoma cell line, Jurkat cell line membrane bound polysomes,epileptic frontal cortex, primary dendritic cells, and a number ofothers. The novel gene related to prohibitin and BAP-37 (SEQ ID NO:45)may prove quite useful as a diagnostic for tumorigenesis, as well as atarget for therapeutic intervention of such an event. Thus, although theprecise functional role of the prohibitin family members are less thanclear, it is quite likely that such homologues are involved in suchcomplex processes as development, senescence, and tumor suppression.Therefore a novel gene, such as HMWGS46 (SEQ ID NO:16 and NO:31), mayprove quite useful as a diagnostic for tumorigenesis, as well as atarget for therapeutic intervention of such an event.

A human cDNA clone encoding a novel epidermal growth factor receptor(EGFR)-like molecule (SEQ ID NO:32) is also disclosed. The novelEGFR-like cDNA clone (SEQ ID NO:17) presented herein was originallyidentified in an activated human neutrophil cDNA library. The clonecontains a 704 nucleotide insert (SEQ ID NO:17) which encodes a 168amino acid polypeptide (SEQ ID NO:32). A BLAST analysis of the predictedamino acid sequence of HNFGW06 (SEQ ID NO:32) demonstrates that thisnovel clone exhibits approximately 85% identity and 90% similarity to aprotein designated epidermal growth factor receptor-related protein[Homo sapiens] (SEQ ID NO:46). (See FIG. 15.) The expression profile ofthe HNFGW06 clone (SEQ ID NO:17) in the HGS database indicates theexistence of a fairly highly restricted expression pattern. In additionto the activated neutrophil library from which this clone (SEQ ID NO:17)was obtained, it also appears in the following HGS human cDNA libraries:synovial sarcoma, smooth muscle, placenta, and possibly primarydendritic cells.

The novel EGFR-like cDNA clone HNFGW06 (SEQ ID NO:17) may lead to anumber of exciting possibilities for therapeutic and/or diagnostictreatments or reagents. For example, HNFGW06 (SEQ ID NO:17 and NO:32)may be involved in the onset of human breast cancers as well. Inaddition, due to the fact that TGF-a acts through binding to the EGFR(SEQ ID NO:46), it is possible that HNFGW06 (SEQ ID NO:17 and NO:32) mayalso play a role in a variety of gastric processes including regulationof acid secretion, regulation of mucous cell growth, and protectionagainst ethanol- and aspirin-induced injury to gastric tissues.

Generating Polynucleotides

Polynucleotides of the present invention encoding a receptor may beobtained using standard cloning and screening, from a cDNA libraryderived from mRNA in cells specified in Table 1 using the expressedsequence tag (EST) analysis (Adams, M. D., et al. Science (1991)252:1651-1656; Adams, M. D. et al., Nature, (1992) 355:632-634; Adams,M. D., et al., Nature (1995) 377 Supp:3-174.) Polynucleotides of theinvention can also be obtained from natural sources such as genomic DNAlibraries or can be synthesized using well known and commerciallyavailable techniques.

The nucleotide sequence encoding a receptor polypeptide of SEQ ID NO:Ymay be identical to the polynucleotide encoding SEQ ID NO:Y, or it maybe a sequence, which as a result of the redundancy (degeneracy) of thegenetic code, also encodes the polypeptide of SEQ ID NO:Y.

When the polynucleotides of the invention are used for the recombinantproduction of a receptor polypeptide, the polynucleotide may include thecoding sequence for the mature polypeptide or a fragment thereof, byitself; the coding sequence for the mature polypeptide or fragment inreading frame with other coding sequences, such as those encoding aleader or secretory sequence, a pre-, or pro- or prepro-proteinsequence, or other fusion peptide portions. For example, a markersequence which facilitates purification of the fused polypeptide can beencoded. In certain preferred embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., ProcNatl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotidemay also contain non-coding 5′ and 3′ sequences, such as transcribed,non-translated sequences, splicing and polyadenylation signals, ribosomebinding sites and sequences that stabilize mRNA.

Further preferred embodiments are polynucleotides encoding receptorvariants comprising the amino acid sequence of receptor polypeptide ofTable 1 (SEQ ID NO:Y) in which several, 5-10, 1-5, 1-3, 1-2 or 1 aminoacid residues are substituted, deleted or added, in any combination.

The present invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the presentinvention especially relates to polynucleotides which hybridize understringent conditions to the herein above-described polynucleotides. Asherein used, the term “stringent conditions” means hybridization willoccur only if there is at least 80%, and preferably at least 90%, andmore preferably at least 95%, yet even more preferably 97-99% identitybetween the sequences.

Polynucleotides of the invention, which are identical or sufficientlyidentical to a nucleotide sequence contained in SEQ ID NO:X or afragment thereof, or to the cDNA insert in the plasmid deposited at theATCC™, or a fragment thereof, may be used as hybridization probes forcDNA and genomic DNA, to isolate full-length cDNAs and genomic clonesencoding the receptor and to isolate cDNA and genomic clones of othergenes (including genes encoding homologs and orthologs) that have a highsequence similarity to the receptor gene. Such hybridization techniquesare known to those of skill in the art. Typically these nucleotidesequences are 80% identical, preferably 90% identical, more preferably95% identical to that of the referent. The probes generally willcomprise at least 15 nucleotides. Preferably, such probes will have atleast 30 nucleotides and may have at least 50 nucleotides. Particularlypreferred probes will range between 30 and 50 nucleotides.

In one embodiment, to obtain a polynucleotide encoding the receptorpolypeptide, including homologs and orthologs from other species,comprises the steps of screening an appropriate library under stringenthybridization conditions with a labeled probe having the SEQ ID NO:X ora fragment thereof; and isolating full-length cDNA and genomic clonescontaining said polynucleotide sequence. Such hybridization techniquesare well known to those of skill in the art. Stringent hybridizationconditions are as defined above or, alternatively, conditions underovernight incubation at 42° C. in a solution comprising: 50% formamidc,5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate(pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 microgram/mldenatured, sheared salmon sperm DNA, followed by washing the filters in0.1×SSC at about 65° C.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to animal and human disease.

Vectors, Host Cells, Expression

The present invention also relates to vectors which comprise apolynucleotide or polynucleotides of the present invention, and hostcells which are genetically engineered with vectors of the invention andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof for polynucleotidesof the present invention. Introduction of polynucleotides into hostcells can be effected by methods described in many standard laboratorymanuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY (1986)and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)such as calcium phosphate transfection, DEAE-dextran mediatedtransfection, transvection, microinjection, cationic lipid-mediatedtransfection, electroporation, transduction, scrape loading, ballisticintroduction or infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, Streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanomacells; and plant cells.

A great variety of expression systems can be used. Such systems include,among others, chromosomal, episomal and virus-derived systems, e.g.,vectors derived from bacterial plasmids, from bacteriophage, fromtransposons, from yeast episomes, from insertion elements, from yeastchromosomal elements, from viruses such as baculoviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression systems may contain control regions that regulate as well asengender expression. Generally, any system or vector suitable tomaintain, propagate or express polynucleotides to produce a polypeptidein a host may be used. The appropriate nucleotide sequence may beinserted into an expression system by any of a variety of well-known androutine techniques, such as, for example, those set forth in Sambrook etal., MOLECULAR CLONING, A LABORATORY MANUAL (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the desired polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

If the receptor polypeptide is to be expressed for use in screeningassays, generally, it is preferred that the polypeptide be produced atthe surface of the cell. In this event, the cells may be harvested priorto use in the screening assay. If the receptor polypeptide is secretedinto the medium, the medium can be recovered in order to recover andpurify the polypeptide; if produced intracellularly, the cells mustfirst be lysed before the polypeptide is recovered.

Receptor polypeptides can be recovered and purified from recombinantcell cultures by well-known methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography is employed for purification. Well known techniques forrefolding proteins may be employed to regenerate active conformationwhen the polypeptide is denatured during isolation and or purification.

Diagnostic Assays

This invention also relates to the use of receptor polynucleotides orpolypeptides for use as diagnostic reagents. Detection of a mutated formof the receptor gene associated with a dysfunction will provide adiagnostic tool that can add to or define a diagnosis of a disease orsusceptibility to a disease which results from under-expression,over-expression or altered expression of the receptor. Individualscarrying mutations in the receptor gene may be detected at the DNA levelby a variety of techniques.

Nucleic acids for diagnosis may be obtained from a subject's cells, suchas from blood, urine, saliva, tissue biopsy or autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR or other amplification techniques prior toanalysis. RNA or cDNA may also be used in similar fashion. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to labeled receptor nucleotide sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNasc digestion or by differences in melting temperatures.DNA sequence differences may also be detected by alterations inelectrophoretic mobility of DNA fragments in gels, with or withoutdenaturing agents, or by direct DNA sequencing. (See, e.g., Myers etal., Science (1985) 230:1242.) Sequence changes at specific locationsmay also be revealed by nuclease protection assays, such as RNase and S1protection or the chemical cleavage method. (See Cotton et al., ProcNatl Acad Sci USA (1985) 85: 4397-4401.) In another embodiment, an arrayof oligonucleotides probes comprising receptor nucleotide sequence orfragments thereof can be constructed to conduct efficient screening ofe.g., genetic mutations. Array technology methods are well known andhave general applicability and can be used to address a variety ofquestions in molecular genetics including gene expression, geneticlinkage, and genetic variability. (See for example: M. Chee et al.,Science, Vol 274, pp 610-613 (1996).)

The diagnostic assays offer a process for diagnosing or determining asusceptibility to specific diseases through detection of mutation in thereceptor gene by the methods described.

In addition, specific diseases can be diagnosed by methods comprisingdetermining from a sample derived from a subject an abnormally decreasedor increased level of receptor polypeptide or receptor mRNA. Decreasedor increased expression can be measured at the RNA level using any ofthe methods well known in the art for the quantitation ofpolynucleotides, such as, for example, PCR, RT-PCR, RNase protection,Northern blotting and other hybridization methods. Assay techniques thatcan be used to determine levels of a protein in a sample derived from ahost are well-known to those of skill in the art. Such assay methodsinclude radioimmunoassays, competitive-binding assays, Western Blotanalysis and ELISA assays.

Thus in another aspect, the present invention relates to a diagnostickit for a disease or susceptibility to a disease which comprises:

(a) a receptor polynucleotide, preferably the nucleotide sequence of SEQID NO:X, or a fragment thereof;

(b) a nucleotide sequence complementary to that of (a);

(c) a receptor polypeptide, preferably the polypeptide of SEQ ID NO:Y,or a fragment thereof; or

(d) an antibody to a receptor polypeptide, preferably to the polypeptideof SEQ ID NO: Y.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component.

Chromosome Assays

The nucleotide sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. The mapping of relevant sequences to chromosomes accordingto the present invention is an important first step in correlating thosesequences with gene associated disease. Once a sequence has been mappedto a precise chromosomal location, the physical position of the sequenceon the chromosome can be correlated with genetic map data. Such data arefound, for example, in V. McKusick, Mendelian Inheritance in Man(available on line through Johns Hopkins University Welch MedicalLibrary). The relationship between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (coinheritance of physically adjacent genes).

The differences in the cDNA or genomic sequence between affected andunaffected individuals can also be determined. If a mutation is observedin some or all of the affected individuals but not in any normalindividuals, then the mutation is likely to be the causative agent ofthe disease.

Antibodies

The polypeptides of the invention or their fragments or analogs thereof,or cells expressing them can also be used as immunogens to produceantibodies immunospecific for the receptor polypeptides. The term“immunospecific” means that the antibodies have substantially greateraffinity for the polypeptides of the invention than their affinity forother related polypeptides in the prior art.

Antibodies generated against the receptor polypeptides can be obtainedby administering the polypeptides or epitope-bearing fragments, analogsor cells to an animal, preferably a nonhuman, using routine protocols.For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler, G. and Milstein, C.,Nature (1975) 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., Immunology Today (1983) 4:72) andthe EBV-hybridoma technique (Cole et al., MONOCLONAL ANTIBODIES ANDCANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can also be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms including other mammals, may be used to express humanizedantibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides byaffinity chromatography.

Antibodies against receptor polypeptides may also be employed to treatdiseases.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in a mammal which comprises inoculating themammal with a receptor polypeptide, or a fragment thereof, adequate toproduce antibody and/or T cell immune response to protect said animalfrom a disease. Yet another aspect of the invention relates to a methodof inducing immunological response in a mammal which comprises,delivering a receptor polypeptide via a vector directing expression ofthe receptor polynucleotide in vivo in order to induce such animmunological response to produce antibody to protect said animal fromdiseases.

Further aspect of the invention relates to an immunological/vaccineformulation (composition) which, when introduced into a mammalian host,induces an immunological response in that mammal to a receptorpolypeptide wherein the composition comprises a receptor polypeptide orreceptor gene. The vaccine formulation may further comprise a suitablecarrier. Since a receptor polypeptide may be broken down in the stomach,it is preferably administered parenterally (including subcutaneous,intramuscular, intravenous, intradermal etc. injection). Formulationssuitable for parenteral administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation instonic with theblood of the recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents or thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample, sealed ampoules and vials and may be stored in a freeze-driedcondition requiring only the addition of the sterile liquid carrierimmediately prior to use. The vaccine formulation may also includeadjuvant systems for enhancing the immunogenicity of the formulation,such as oil-in water systems and other systems known in the art. Thedosage will depend on the specific activity of the vaccine and can bereadily determined by routine experimentation.

Screening Assays

The receptor polypeptide of the present invention may be employed in ascreening process for compounds which bind the receptor and whichactivate (agonists) or inhibit activation of (antagonists) the receptorpolypeptide of the present invention. Thus, polypeptides of theinvention may also be used to assess the binding of small moleculesubstrates and ligands in, for example, cells, cell-free preparations,chemical libraries, and natural product mixtures. These substrates andligands may be natural substrates and ligands or may be structural orfunctional mimetics. See Coligan et al., Current Protocols in Immunology1(2):Chapter 5 (1991).

The receptor polypeptides are responsible for many biological functions,including many pathologies. Accordingly, it is desirous to findcompounds and drugs which stimulate the receptor on the one hand andwhich can inhibit the function of the receptor on the other hand. Ingeneral, agonists are employed for therapeutic and prophylactic purposesfor such conditions and diseases. Antagonists may be employed for avariety of therapeutic and prophylactic purposes for such conditions anddiseases.

In general, such screening procedures involve producing appropriatecells which express the receptor polypeptide of the present invention onthe surface thereof. Such cells include cells from mammals, yeast,Drosophila or E. coli. Cells expressing the receptor (or cell membranecontaining the expressed receptor) are then contacted with a testcompound to observe binding, or stimulation or inhibition of afunctional response.

The assays may simply test binding of a candidate compound whereinadherence to the cells bearing the receptor is detected by means of alabel directly or indirectly associated with the candidate compound orin an assay involving competition with a labeled competitor. Further,these assays may test whether the candidate compound results in a signalgenerated by activation of the receptor, using detection systemsappropriate to the cells bearing the receptor at their surfaces.Inhibitors of activation are generally assayed in the presence of aknown agonist and the effect on activation by the agonist by thepresence of the candidate compound is observed.

Further, the assays may simply comprise the steps of mixing a candidatecompound with a solution containing a receptor polypeptide to form amixture, measuring receptor activity in the mixture, and comparing thereceptor activity of the mixture to a standard.

The receptor cDNA, protein and antibodies to the protein may also beused to configure assays for detecting the effect of added compounds onthe production of receptor mRNA and protein in cells. For example, anELISA may be constructed for measuring secreted or cell associatedlevels of receptor protein using monoclonal and polyclonal antibodies bystandard methods known in the art, and this can be used to discoveragents which may inhibit or enhance the production of the receptor (alsocalled antagonist or agonist, respectively) from suitably manipulatedcells or tissues. Standard methods for conducting screening assays arewell understood in the art.

Examples of potential receptor antagonists include antibodies or, insome cases, oligonucleotides or proteins which are closely related tothe ligand of the receptor, e.g., a fragment of the ligand, or smallmolecules which bind to the receptor but do not elicit a response, sothat the activity of the receptor is prevented.

Thus in another aspect, the present invention relates to a screening kitfor identifying agonists, antagonists, ligands, receptors, substrates,enzymes, etc. for receptor polypeptides; or compounds which decrease orenhance the production of receptor, which comprises:

(a) a receptor polypeptide, preferably that of SEQ ID NO:Y;

(b) a recombinant cell expressing a receptor polypeptide, preferablythat of SEQ ID NO:Y;

(c) a cell membrane expressing a receptor polypeptide; preferably thatof SEQ ID NO: Y; or

(d) antibody to a receptor polypeptide, preferably that of SEQ ID NO: Y.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component.

Prophylactic and Therapeutic Methods

This invention provides methods of treating an abnormal conditionsrelated to both an excess of and insufficient amounts of receptoractivity.

If the activity of the receptor is in excess, several approaches areavailable. One approach comprises administering to a subject aninhibitor compound (antagonist) as described along with apharmaceutically acceptable carrier in an amount effective to inhibitactivation by blocking the binding of ligands to the receptor or byinhibiting a second signal, and thereby alleviating the abnormalcondition.

In another approach, soluble forms of the receptor polypeptides stillcapable of binding the ligand in competition with endogenous receptormay be administered. Typical embodiments of such competitors comprisefragments of the receptor polypeptide.

In still another approach, expression of the gene encoding endogenousreceptor can be inhibited using expression blocking techniques. Knownsuch techniques involve the use of antisense sequences, eitherinternally generated or separately administered. (See, for example,O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988).) Alternatively, oligonucleotides which form triple helices withthe gene can be supplied. (See, for example, Lee et al., Nucleic AcidsRes (1979) 6:3073; Cooney et al., Science (1988) 241:456; Dervan et al.,Science (1991) 251:1360.) These oligomers can be administered per se orthe relevant oligomers can be expressed in vivo.

For treating abnormal conditions related to an under-expression of thereceptor and its activity, several approaches are also available. Oneapproach comprises administering to a subject a therapeuticallyeffective amount of a compound which activates the receptor, i.e., anagonist as described above, in combination with a pharmaceuticallyacceptable carrier, to thereby alleviate the abnormal condition.Alternatively, gene therapy may be employed to effect the endogenousproduction of the receptor by the relevant cells in the subject. Forexample, a polynucleotide of the invention may be engineered forexpression in a replication defective retroviral vector, as discussedabove. The retroviral expression construct may then be isolated andintroduced into a packaging cell transduced with a retroviral plasmidvector containing RNA encoding a polypeptide of the present inventionsuch that the packaging cell now produces infectious viral particlescontaining the gene of interest. These producer cells may beadministered to a subject for engineering cells in vivo and expressionof the polypeptide in vivo. For overview of gene therapy, see Chapter20, Gene Therapy and other Molecular Genetic-based TherapeuticApproaches, (and references cited therein) in Human Molecular Genetics,T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996).

Formulation and Administration

Peptides, such as the soluble form of receptor polypeptides, andagonists and antagonist peptides or small molecules, may be formulatedin combination with a suitable pharmaceutical carrier. Such formulationscomprise a therapeutically effective amount of the polypeptide orcompound, and a pharmaceutically acceptable carrier or excipient. Suchcarriers include but are not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof.Formulation should suit the mode of administration, and is well withinthe skill of the art. The invention further relates to pharmaceuticalpacks and kits comprising one or more containers filled with one or moreof the ingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds. Preferred forms of systemic administration of thepharmaceutical compositions include injection, typically by intravenousinjection. Other injection routes, such as subcutaneous, intramuscular,or intraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if properly formulated in enteric or encapsulatedformulations, oral administration may also be possible. Administrationof these compounds may also be topical and/or localized, in the form ofsalves, pastes, gels and the like.

The dosage range required depends on the choice of peptide, the route ofadministration, the nature of the formulation, the nature of thesubject's condition, and the judgment of the attending practitioner.Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject.Wide variations in the needed dosage, however, are to be expected inview of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art.

Polypeptides used in treatment can also be generated endogenously in thesubject, in treatment modalities often referred to as “gene therapy” asdescribed above. Thus, for example, cells from a subject may beengineered with a polynucleotide, such as a DNA or RNA, to encode apolypeptide ex vivo, and for example, by the use of a retroviral plasmidvector. The cells are then introduced into the subject.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

1. An isolated polynucleotide comprising a nucleotide sequence that hasat least 80% identity over its entire length to a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding the polypeptide of SEQ ID NO:Y; (b) a nucleotide sequencehaving at least 80% identity to a nucleotide sequence encoding thepolypeptide expressed by the cDNA insert deposited at the ATCC; or (c) anucleotide sequence complementary to said isolated polynucleotide. 2.The polynucleotide of claim 1, wherein said polynucleotide is (a). 3.The polynucleotide of claim 1, wherein said polynucleotide is (b). 4.The polynucleotide of claim 1, wherein said polynucleotide is (c).
 5. Amethod of diagnosing a disease or a susceptibility to a disease in asubject that is related to the presence of mutations in, or theproduction of, a nucleotide sequence, comprising collecting a samplefrom a subject and: (a) determining the presence or absence of amutation in a nucleotide sequence encoding the polypeptide of SEQ IDNO:Y in the genome of said subject; and/or (b) analyzing for thepresence or amount of the nucleotide in said sample derived from saidsubject.
 6. The method of claim 5, wherein the polynucleotide is afragment of the nucleotide sequence encoding the polypeptide of SEQ IDNO:Y.
 7. The method of claim 6, wherein the polynucleotide fragmentcomprises at least 30 consecutive nucleotides of the nucleotide sequenceencoding the polypeptide of SEQ ID NO:Y
 8. The method of claim 6,wherein the polynucleotide fragment comprises at least 50 consecutivenucleotides of the nucleotide sequence encoding the polypeptide of SEQID NO:Y.
 9. An isolated polypeptide comprising an amino acid sequencethat has at least 80% identity over its entire length to an amino acidsequence encoding the polypeptide of SEQ ID NO:Y.
 10. The polypeptide ofclaim 9, wherein the polypeptide comprises at least 30 consecutive aminoacids of SEQ ID NO:Y.
 11. The polypeptide of claim 9, wherein thepolypeptide comprises at least 50 consecutive amino acids of SEQ IDNO:Y.